Figure 1.

Crosslinking of pre-tRNA and RNase P holoenzyme reconstituted with AzP-labeled S49C P protein. The RNase P⋅pre-tRNA complex was formed by incubating ³²P-labeled pre-tRNA (5–10 nM pTR14 RNA) and 0.4 μM holoenzyme at 37°C in 50 mM Mes-Tris buffer, pH 6.0, 5 mM CaCl₂. Samples were exposed to UV light (312 nm) for 1 min. The crosslinked (XL) products were separated on 15% SDS/PAGE and visualized by using a PhosphorImager. (A) Lane 1, pre-tRNA alone; lane 2, pre-tRNA and P RNA; lane 3, pre-tRNA and holoenzyme; lane 4, pre-tRNA and holoenzyme reconstituted with S49C P protein; lanes 5–7, pre-tRNA and holoenzyme reconstituted with AzP-labeled S49C P protein, no irradiation (lane 5), 1-min irradiation (lane 6), and irradiation followed by proteinase K digestion (five units at 37°C for 20 min) (lane 7). (B) Crosslinking depends on the length of the leader. Lane 1, mature tRNA; lanes 2–9, pre-tRNA substrates with a leader sequence of 2, 3, 4, 5, 6, 7, 10, and 14 nt, respectively. (C) RNA specificity of crosslinks. pTR14 and holoenzyme reconstituted with AzP-S49C P protein was irradiated in the absence of competitor (lane 1), or in the presence of 5 μM mature tRNA (lane 2), 5 μM 5′ leader RNA (lane 3), 250 μg/ml of poly G RNA (lane 4), 250 μg/ml of poly C RNA (lane 5), and 2 μM pTR14 RNA (lane 6) as competitors. Only the substrate effectively inhibits formation of the crosslink. (D) Cleavage of the crosslinked pTR14/holoenzyme complex. The cleavage of pTR14 substrate crosslinked to AzP-S49C RNase P was initiated by the addition of MgCl₂ (10 mM final concentration) and unlabeled pTR14 substrate (10 μM final concentration) to inhibit the activity of the uncrosslinked RNase P. The reaction was incubated at 37°C, and a 5-μl aliquot was quenched at defined times by the addition of SDS sample buffer. The crosslinked pre-tRNA (XL) and free pre-tRNA were separated on a 15% SDS/PAGE gel and quantitated by using a PhosphorImager. Lanes 1 and 2, ³²P-labeled pTR14 and holoenzyme reconstituted with AzP-S49C P protein before and after UV irradiation; lanes 3–9, 8, 15, 21, 27, 34, 40, and 300 sec, respectively, after the addition of MgCl₂. (E) Single turnover cleavage of pTR14 catalyzed by RNase P reconstituted with unmodified S49C P protein. ³²P-labeled pre-tRNA (10 nM pTR14) was incubated with a saturating concentration (0.4 μM) of RNase P at 37°C in 50 mM Mes, 50 mM Tris, pH 6.0, 5 mM CaCl₂, and the cleavage reaction was initiated by the addition of MgCl₂ (10 mM final concentration). Pre-tRNA (S) and the 5′ leader sequence (P) were separated on a 15% SDS/PAGE gel and quantitated by using a PhosphorImager. Lane 1, ³²P-labeled pTR14 and holoenzyme before addition of MgCl₂; lanes 2–9, 6, 12, 18, 23, 30, 36, 43, and 314 sec, respectively, after the addition of MgCl₂. (F) The percent substrate as a function of time is plotted for the single turnover cleavage of pTR14 catalyzed by wild-type RNase P (□), RNase P reconstituted with S49C P protein (▵), and pTR14 crosslinked to RNase P reconstituted with AzP-S49C P protein (•). The data are fit to a single exponential decay by using the Kaleidagraph (Synergy software) curve-fitting program; the fitted rate constant and endpoint are 0.09 ± 0.01 s⁻¹ and 6 ± 2% for wild-type RNase P and 0.05 ± 0.01 s⁻¹ and 5 ± 2% for either RNase P reconstituted with S49C P protein or pTR14 crosslinked to RNase P reconstituted with AzP-S49C P protein.